Because the pathogenesis of enterovirus 71 (EV71) remains mostly ambiguous, identifying the factors that mediate viral binding and entry to host cells is indispensable to ultimately uncover the mechanisms that underlie
virus infection and pathogenesis. Despite the identification of several receptors/attachment molecules for EV71, the binding, entry, and
infection mechanisms of EV71 remain unclear. Herein, we employed glycoproteomic approaches to identify human
nucleolin as a novel binding receptor for EV71.
Glycoproteins purified by
lectin chromatography from the membrane extraction of human cells were treated with
sialidase, followed by immunoprecipitation with EV71 particles. Among the 16
proteins identified by tandem mass spectrometry analysis, cell surface
nucleolin attracted our attention. We found that EV71 interacted directly with
nucleolin via the VP1
capsid protein and that an antinucleolin antibody reduced the binding of EV71 to human cells. In addition, the knockdown of cell surface
nucleolin decreased EV71 binding,
infection, and production in human cells. Furthermore, the expression of human
nucleolin on the cell surface of a mouse cell line increased EV71 binding and conferred EV71
infection and production in the cells. These results strongly indicate that human
nucleolin can mediate EV71 binding to and
infection of cells. Our findings also demonstrate that the use of glycoproteomic approaches is a reliable methodology to discover novel receptors for pathogens.
IMPORTANCE: Outbreaks of EV71 have been reported in Asia-Pacific countries and have caused thousands of deaths in young children during the last 2 decades. The discovery of new EV71-interacting molecules to understand the
infection mechanism has become an emergent issue. Hence, this study uses glycoproteomic approaches to comprehensively investigate the EV71-interacting
glycoproteins. Several EV71-interacting
glycoproteins are identified, and the role of cell surface
nucleolin in mediating the attachment and entry of EV71 is characterized and validated. Our findings not only indicate a novel target for uncovering the EV71
infection mechanism and anti-EV71
drug discovery but also provide a new strategy for
virus receptor identification.